Method for preparing human meloma vaccine

ABSTRACT

There is disclosed a method of preparing a vaccine suitable for administration to humans for the prevention or treatment of cancer. The vaccine is prepared by culturing human cancer cells in a serum-free medium and recovering from the culture medium the cell surface antigens shed from the human cancer cells during culturing. After purification, the collected or recovered shed antigens are employed to produce a vaccine consisting essentially of said antigens for the treatment or prevention of human cancer.

This is a divisional application of application Ser. No. 41,864 filedApr. 23, 1987 abandoned in favor of its continuation application Ser.No. 485,780 filed Feb. 22, 1990, now U.S. Pat. No. 5,030,621 issued Jul.9, 1991.

BACKGROUND OF THE INVENTION

This invention relates to human vaccines, such as vaccines forprotection against pathogenic microorganisms, e.g. bacterial infectionsand the like, and to human anti-cancer vaccines. More particularly, andin one special embodiment, this invention relates to the preparation ofhuman anti-cancer vaccines useful for the prevention and/or treatment ofcancer, such as melanoma, breast cancer, colon cancer, lung cancer andother such cancers.

For the treatment of cancer, it has been suggested to increase tumorprotective immunity by active immunization to tumor antigens, see (1)the article by R. K. Oldham entitled "Biologicals and BiologicalResponse Modifiers: Fourth Modality of Cancer Treatment", Cancer TreatRep (1984);68:221-232. and (2) the article by M. J. Mastrangelo et alentitled "Current Condition and Prognosis of Tumor Immuunotherapy: ASecond Opinion", Cancer Treat Rep (1984);68:207-219. Unfortunately, thisapproach for the prevention and/or treatment of cancer has not beensuccessful or completely satisfactory because of a number of problems,such as the absence in the vaccine of tumor antigens expressed by thetumor to be treated, poor characterization of the antigens in tumorvaccines, the contamination of vaccines by immunogenic but undesirablematerial, such as fetal calf serum (FCS) protein or transplantationantigens and additionally due to the antigenic heterogenicity of thecancer cells. Moreover, such tumor vaccines were often prepared fromfresh tumor cells, the supply of which is limited so that the propertiesof the vaccines are not reproducible.

A clinical trial was conducted to evaluate the toxicity andimmunogenicity in man of a partially purified, polyvalent, melanomaantigen vaccine and some success was indicated, see the abstract of thepaper by J. C. Bystryn et al published by The Society for InvestigativeDermatology, Inc. entitled "Phase 1 Trial of Specific Immunotherapy ofMelanoma with a Polyvalent Melanoma Antigen Vaccine"

It is an object of this invention to provide an improved anti-cancervaccine.

It is another object of this invention to provide a technique for thepreparation of an improved anti-cancer vaccine.

It is another object of this invention to provide an immunotherapy forthe prevention and/or treatment of human cancer.

Yet another object of this invention is to provide a technique for theproduction of reproducible anti-cancer vaccines.

Still another object of this invention is to provide a technique for thepreparation of an anti-cancer vaccine useful when introduced into apatient to prevent and/or to treat cancer.

It is still another object of this invention to provide a technique forthe preparation of vaccines for diseases caused by infectious cellularand/or subcellular organisms and/or viruses.

It is yet another object of this invention to provide a technique forthe preparation of clinically or biologicall important material shedfrom the surface of cells and the like.

How these and other objects of this invention are achieved will becomeapparent in the light of the accompanying disclosure. In at least oneembodiment of the practices of this invention at least one of theforegoing objects will be achieved.

SUMMARY OF THE INVENTION

A human vaccine useful for the prevent-ion and/or treatment ofinfections caused by pathogenic microorganisms, including viral, fungal,protozoal, amoebic and bacterial infections and the like, human cancer,including human melanoma, a skin cancer, human lung cancer, human breastcancer, human colon cancer and other human cancers is produced. For theproduction of a human cancer vaccine, the vaccine is produced byculturing human cancer cells, such as human melanoma cells, in aserum-free medium for the collection in the medium of cancer antigens,such as multiple melanoma associated antigens (MAAs). The vaccineproduced from the shed material contains multiple cell surface antigensincluding tumor antigens and if prepared from cells adapted to and grownin a serum-free medium, is free of calf serum proteins. The use ofbioreactors for the bulk culture of the cells and the like for vaccineproduction is particularly useful.

The vaccine is employed for the prevention and/or treatment of cancer inhumans by administering the vaccine into the extremities of the patienta number of times a month and then once every few months thereafter foran extended period of time, such as 1-4 years, more or less. Asindicated, the invention is applicable for the production of vaccinesfor the prevention and treatment of other cancers as well as forinfectious diseases caused by bacteria, fungi, rickettsia, virus andother cellular and subcellular organisms.

DETAILED DESCRIPTION OF THE INVENTION

The practices of this invention are hereinafter described in some detailwith respect to the production of a human melanoma antigen vaccine andthe treatment of melanoma patients. As indicated hereinabove, however,the practices of this invention are also applicable to the production ofa human lung cancer vaccine, a human breast cancer vaccine, a humancolon cancer vaccine and other human cancer vaccines as well as vaccinesfor infectious diseases, particularly infectious diseases caused bybacteria, fungi and other microorganisms.

In the preparation of a human melanoma antigen vaccine in accordancewith this invention the vaccine was prepared from material shed by fourlines of human melanoma cells: HM31, HM34, HM49, and SK-Mel-28. Thecells were selected on the basis of the cells expressing differentpatterns of cell surface melanoma antigens and adapted to and maintainedin serum-free medium, see the article by J. P. Mather et al entitled"The Growth of Mouse Melanoma Cells in Hormone Supplemented Serum-FreeMedium", Exp Cell Res 1979;120:191-200, for at least 8 weeks prior touse.

Antisera

A panel of eight murine monoclonal antibodies and two rabbit polyclonalmelanoma antisera were used for MAA immunophenotyping. The antigensdefined are listed in accompanying Table 1. The antigens defined by themurine and rabbit antisera are different, even though some may havesimilar molecular weights, as shown by variations in their distributionamong melanomas (Table 3).

Vaccine Preparation

Melanoma cells were incubated at a concentration of 2×10⁶ /ml serum-freeRPMI 1640 medium. After 3 hours at 37° C., the medium was collected andthe cells were removed by centrifugation at 2,000 g for 10 minutes, andlarger particles were removed by recentrifugation at 12,000 g for 15minutes. Equal volumes of medium from the four cells lines were pooled,concentrated 10-fold by vaccum ultrafiltration and made up to a finalconcentration. In some cases vaccines were prepared with furthertreatment including the addition of a non-ionic surfactant, e.g. 0.5%Nonidet P-40 (NP-40) and 0.02% sodium azide as a preservative, and thenultracentrifuged at 100,000 g for 90 minutes. The supernatant wasdialyzed at 4° C. against normal saline with 0.02% sodium azide and madeup to the desired protein concentration by the addition of normalsaline, passed through a 0.1 um Millex Millipore filter to removemicroorganisms; and 0.5 ml aliquots dispensed into sterile, pyrogen-freeglass vials. The vials were stored at -70° C. until used.

A control vaccine was similarly prepared from a pool of normalperipheral leukocytes obtained from five normal individuals. Prior touse, the vaccine was tested for aerobic and anerobic bacteria, fungi,and hepatitis antigen and also tested for mycoplasma by bisbenzamide DNAfluorochrome stain and for pyrogens by the limulus test. For MAAphenotyping, the vaccine was prepared from cells radioiodinated by thelactoperoxidase technique, see the article by J. C. Bystryn et alentitled "Identification and Solubilization of Iodinated Cell SurfaceHuman Melanoma Associated Antigens", Int J Cancer 1977;20:165-172, andradiolabeled cells were lysed in 10 ml of 0.15 NP-40.

Assays

Protein concentration was measured by the Lowry method, see the articleby O. H. Lowry et al entitled "Protein Measurement with the FolinphenolReagent", J Biol Chem 1951;193:265-275. Profiles of proteins wereanalyzed by SDS-PAGE and silver staining or autoradiography, see thearticles, respectively, by J. C. Bystryn entitled "Comparison ofCell-Suface Human Melanoma-Associated Antigens Identified by Rabbit andMurine Antibodies", Hybridoma 1982;4:465-472. and B. R. Oakley et al "ASimplified Ultrasensitive Silver Stain for Detecting Proteins inPolyacrylamide Gels", Anal Biochem 1980;105:361-363. Radioactivityassociated with labeled macromolecules was measured by precipitationwith 10% trichloroacetic acid. MAAs were assayed by protein A-sepharoseimmunoprecipitation of labeled antigens by using the panel of antiseralisted in Table 1, followed by SDS-PAGE and autoradiography. FCSproteins and Dr antigens were assayed in a similar manner by usingappropriate antisera.

Clinical Studies

The vaccine was used to immunize 13 patients with metastatic malignantmelanoma, see accompanying Table 4. All had intact immune function, asevidenced by skin reactivity to at least one standard recall antigen orby the ability to be sensitized to dinitrochlorobenzene (DNCB). A singlelot of vaccine was used to treat all patients. Therapy was initiated atleast 1 month following surgery, chemotherapy, or radiation therapy. Noother therapy was given concurrently, and 0.1 ml of vaccine wasadministered without adjuvant into each of the four extremities weeklyfor eight weeks, monthly for three months, then twice at three monthintervals and thereafter every six months. The initial dose of 0.25ug/site was increased every 2 weeks to 1.0, 10.0 and 50 ug/site.Physical examination, blood count, sedimentation rate, serum chemistryprofile, urinalysis, serum immunoelectrophoresis, immune complex levels,and chest x-ray were performed prior to therapy, twice monthly and every2 months thereafter. Chest x-rays were repeated every 3 months and otherradiological studies as indicated. Serum for melanoma antibody wascollected on two occasions prior to therapy and prior to eachvaccination.

Immune Responses to Melanoma

Antibodies to melanoma surface antigens were assayed by indirectimmunoprecipitation, using as antigen detergent extracts oflactoperoxidase radiodinated melanoma cells see the article by G. K.Naughton et al entitled "Antibodies to Normal Human Melanocytes inVitiligo", J Exp Med 1983;158:246-251. The cells were those used forvaccine production. Each patient served as his own control. Immunizationwas considered to have increased melanoma antibody level if the countsper minute (cpm) immunoprecipitated by postimmune sera were at least 50%greater than the average cpm immunoprecipitated by two preimmune sera inthe same patient. The antigens defined by antibody in selected patientswere identified by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and autoradiography, see reference 11hereinabove. Antibodies of FCS proteins in rabbit and human sera wereassayed by protein A-sepharose immunoprecipitation using fetal calfserum radioiodinated with immobilized lactoperoxidase, see the articleby G. S. David entitled "Protein Iodination with Solid StateLactoperoxidase", Biochemistry 1974;13:1014-1021.

Cellular immunity to melanoma was evaluated by delayed-typehypersensitivity (DTH) reactions to skin tests with 10 ug of vaccine.These were considered positive if induration at least 10 mm in averagediameter persisted for 24-48 hours.

Protein Profile of Vaccine

At least 20 proteins ranging in MW 30 Kd to 70 Kd were present in thevaccine as indicated by SDS-PAGE profile of proteins stained with silverin the material shed by pooled melanoma cells. Additional proteins withMWs ranging to 200 Kd could be visualized in vaccine prepared fromradioiodinated cells upon SDS-PAGE profile of MAAs in vaccine preparedfrom radioiodinated cells immunoprecipitated by monoclonal antibodies.

To minimize the presence of FCS proteins in the vaccine, the vaccine wasprepared from cells maintained in serum-free medium for at least 8weeks. Some experiments confirmed that no detectable FCS proteinspersisted in the vaccine. None could be detected by immunoprecipitationwith anti-FCS serum in vaccine prepared from lactoperoxidaseradioiodinated cells, even though this procedure is sensitive enough todetect FCS adhering to and shed by melanoma cells grown in the presenceof serum, see the article by J. C. Bystryn et al entitled "Release ofSurface Macromolecules by Human Melanoma and Normal Cells", Cancer Res1981;41:910-4. Two rabbits repeatedly immunized to the vaccine developedantibodies to a number of melanoma antigens in the vaccine but not toradioiodinated FCS proteins. By contrast, antibodies to FCS were readilydetected in two control rabbits immunized in parallel to vaccinecontaminated with trace amounts of xenogeneic serum. Finally, noantibodies to FCS were found in 13 patients repeatedly immunized to thevaccine.

To separate MAAs from transplantation antigens, shed material wastreated with 0.5% NP-40 to solubilize aggregates and was subsequentlyultracentrifuged. The effects of this procedure on proteins, tumor, andtransplantation antigens are summarized in Table 2. Ultra-centrifugationof shed material not treated with detergent removed much of theradioactivity associated with labeled macromolecules, in addition tothose MAAs defined by polyclonal antiserum SB29 and 100% of thoseassociated with Dr antigens. Ultracentrifugation of detergent-treatedshed material still resulted in a loss of all detectable Dr antigens butreduced the loss of labeled macromolecules to 17% and of MAAs to 30%.Thus, detergent treatment and ultracentrifugation removed Dr antigenswith an acceptable loss in MAAs.

Antigenic Properties of Vaccine

Vaccine prepared from radioiodinated cells was immunophenotyped with apanel of 10 melanoma antisera. The results are summarized in Table 1.Most of the MAAs tested were present in the vaccine. Three batches ofvaccine prepared several months apart all contained the MAAs tested, seeaccompanying Table 1. Upon freeze-thawing three times or storage at -70°C. for four months, there was no loss in antigenic activity.

Distribution of MAAs in Various Melanomas

Because it is desirable that the vaccine contain at least one tumorantigen which will be present on most of the melanoma tumors to betreated, it was examined whether or not the panel of MAAs in the vaccinesatisfied this requirement. Fifteen melanomas were lactoperoxidaseradioiodinated and immunophenotyped for the MAAs present in the vaccine.There were marked differences, see accompanying Table 3, in the patternof MAAs expressed by each melanoma. However, a11 the melanomas expressedseveral of the MAAs present in the vaccine.

Phase I Trial

Thirteen patients with metastatic melanoma were immunized withincreasing doses of vaccine for 2 months, see accompanying Table 4, and10 of these received 2 to 10 additional booster doses during the period5 months to 2 years. No toxicity was observed other than transienturticaria at the site of injection. A similar urticarial reactionoccurred in the skin of each of five patients tested with sterile salinecontaining an amount of NP-40 equal to that present in the vaccine.There was no reaction to skin tests to sterile saline alone.

Additional Clinical Trials

Clinical studies were performed to determine the toxicity of the vaccinein patients with early melanoma and the effect of several immunizationstrategies on immunogenecity. A total of 55 melanoma patients (36 withStage II disease metastatic to regional nodes and 19 patients with StageIII widely disseminated disease) have been treated. There was notoxicity other than transient inflammation at the site of vaccineinjection.

Immune Response to Immunization

Antibodies to surface antigens on melanoma cells, assayed by indirectimmunoprecipitation, increased in five (38%) patients, see accompanyingTable 5. The increase was usually small, although in one patient(patient 1) peak binding activity was 300% greater than the preimmunelevel. Antibody levels increased in some patients within 1 weekfollowing the first immunization, suggesting that a secondary type ofimmune response was elicited. The immunogenic antigens that stimulatedthe production of melanoma antibodies were identified in three patientsby SDS-PAGE and autoradiography. These were proteins with MWs of about75, 85, and 200+ Kd. The larger antigen appeared to be more immunogenic,since all three patients developed antibodies to it, whereas antibodiesto the other two antigens were present in only one patient. None ofthese antigens was related to FCS proteins, since their binding was notblocked by excess cold FCS. Furthermore, after 2 months of immunization,there was no increase in antibodies to FCS in any of the patients, seeaccompanying Table 6.

Cellular immunity to melanoma, evaluated by DTH reaction to skin testswith the vaccine, was induced in four (31%) patients, see accompanyingTable 5. Skin tests were negative prior to immunization in all patients.Three to six immunizations with 10 ug or more of vaccine were requiredto induce a positive reaction. There was no reaction in positivepatients to skin tests with equal amounts of human albumin or to acontrol vaccine prepared from pooled normal peripheral leukocytes.Overall, there was an increase in humoral and/or cellular immuneresponse to melanoma following immunization in eight (62%) of thepatients.

In subsequent clinical trials it was found that antibody and/or cellularimmune responses to melanoma were induced more frequently in Stage II(69% of 36 patients) than in Stage III (53% of 19 patients) disease. Theability of different immunization schedules, alum or pretreatment withlow dose cyclophosphamide, to potentiate immunogenicity was comparedafter 2 months of immunization. Bi-weekly immunization with a fixedintermediate dose of vaccine was more immunogenic than weeklyimmunization with escalating vaccine doses. Alum increased slightly theintensity of cellular responses while pretreatment with cyclophosphamideaugmented slightly both the incidence and intensity of cellular immuneresponses. There was a reciprocal relationship between the induction ofhumoral and cellular immune responses. The most effective immunizationschedule consisted of petreatment with cyclophosphamide which augmentedantibody and/or cellular immune responses to melanoma in 83% ofpatients.

The vaccine is capable of augmenting immune responses to a patient's owntumor. There were examined the density and distribution of lymphocyticinflammatory cells in cutaneous metastases that developed during thecourse of vaccine immunization in 11 patients and comparison was made ina blinded fashion to the lymphocytic infiltrate present in similarcutaneous melanoma metastases removed from 22 non-immunized, randomlyselected patients. Dense cellular infiltrates (15 lymphoid cells/highpower field HPF) were more frequent in tumor nodules of immunizedpatients (10 of 11 nodules, 91%) than in those of control patients (9 of22 nodules, 41%), p 0.01. The cells were more likely to be infiltratingwithin the tumornodules in immunized patients (7 of 11 nodules, 64%)than in control patients (5 of 22 nodules, 23%), p 0.05. The incidenceof lymphoid cells at the periphery of nodules was similar in bothgroups.

These results indicate that immunization to melanoma vaccine can augmentlymphocyte infiltration into tumors in vivo, and suggests thatpolyvalent allogeneic melanoma vaccines can boost a patient's ability tomount an immune response to his own tumor.

Effect of Immunization on Tumor Growth

In the initial Phase I study, two of the 13 immunized patients have donewell, see accompanying Table 4. There was a complete remission in a46-year-old male (patient 10) who developed three disseminated cutaneousmetastasis one year following excision of a melanoma of the right upperback. Two metastases were removed and the third left as a marker. Itincreased in size 30% during the first months of immunotherapy, wasstable for several months, and then slowly decreased in size by 50%. Itwas removed 10 months after onset of immunotherapy and histologicallywas a regressed malignant melanoma with only pigment-laden macrophagesat the site. The patient is currently disease-free, 36 months afteronset of immunotherapy.

There was a long-term stable disease in a 75-year- 6 old woman (patient9) who developed two cutaneous metastases 2 years after excision of amalignant melanoma of the right calf. These were removed, and she wastreated with dimethyltriazenoimidozole carboxamide (DTIC) andactinomycin D. Three years later, six new cutaneous metastases appeared,and she was started on vaccine immunotherapy. Cutaneous metastasesincreased in number and size during the next 4 months until she had 15.She was continued on immunotherapy, and there has been no furtherincrease in size or number of metastases. The largest lesion regressedspontaneously 6 months after onset of immunotherapy. The patientcurrently has stable disease 14 months after onset of immunotherapy.

In other studies involving 55 patients with stage II melanoma, it wasfound that recurrence of melanoma was less common in patients with acellular immune response to the vaccine (35%) than in those with anantibody response (86%) p<0.05 and the disease free interval wasprolonged from about 39 to greater than 100 weeks. These results suggestthat active immunization to the melanoma vaccine can slow theprogression of melanoma in some patients.

Several features of the technique used to prepare the vaccine deserveattention, because they were designed to minimize problems. The vaccinewas prepared from melanoma cells grown in serum-free medium to avoidcontamination with FCS proteins. These proteins are highly immunogenicand responsible for most immune responses induced by human tumorvaccines prepared from cultured cells, see the articles by P. O.Livingston et al entitled "Serological Responses of Melanoma Patients toVaccines Derived from Allogenic Cultured Melanoma Cells", Int J Cancer1983;31:567-575, P. O. Livington et al entitled "Serological Response ofMelanoma Patients to Vaccines Prepared from VSV Lysates of Autologousand Allogenic Melanoma Cells", Cancer 1985;55-713-720 and M. S. Mahaley,Jr. et al entitled "Immunobiology of Primary Intracranial Tumors. Part8: Serological Responses to Active Immunization of Patients withAnaplastic Gliomas", J Neurosurg 1983;59:208-216. This approach appearsto have been successful, since no FCS proteins were detectable in thevaccine by direct assay or by the more sensitive method of looking foran immune response to these proteins in animals or persons immunized toit.

Cultured cells were used to ensure a continued and reproducible sourceof material, a basic requirement for vaccine development, see referencearticle 1. Three batches of vaccine prepared several months apart allcontained the MAAs that were tested for, indicating that vaccinecontaining similar antigens can be made reproducilibly. Quantitativechanges in the amount of each antigen in the vaccine have not beenexcluded.

The vaccine was prepared from a pool of cells, selected because theyexpressed different patterns of surface MAAs. This was done to create apolyvalent vaccine, which is desirable in treating melanoma cells (and,indeed, for treating other cancer cells) that are antigenicallyheterogeneous, see the articles by J. C. Bystryn et al entitled"Immunophenotype of Human Melanoma Cells in Different Metastases",Cancer Res, in press, 1985, M. Y. Yeh et al entitled "Clonal Variationin Expression of a Human Melanoma Antigen Defined by a MonoclonalAntibody", J Immunol 1981;126:1312-7, A. P. Albino et al entitled"Heterogeneity in Surface Antigen and Glycoprotein Expression of CellLines Derived from Different Melanoma Metastases of the Same Patient", JExp Med 1981;154-1764-1778. The vaccine contained multiple MAAs. Most ofthe MAAs tested for were present in the vaccine; it is therefore likelythat the vaccine contains additional MAAs that were not tested for. Atleast one of the MAAs present in the vaccine was expressed by each of 15metastatic melanomas that were immunophenotyped. Thus, a mixture oftumor antigens in the vaccine would be appropriate to circumventantigenic heretogeneity among melanomas.

Shed antigens were used because they are partially purified. They areseparated from the bulk of cytoplasmic components, which are slowlyreleased (<5%) during the short collection period; surface components,however, are released rapidly, see the article by J. C. Bystryn et alentitled "Shedding and Degradation of Cell-Surface Macromolecules andTumor-Associated Antigens by Human Melanoma". In: Reisfeld R. A,,Ferrone S, eds. Melanoma Antigens and Antibodies, New York: PlenumPress. 1982:37-52. In addition, shed material is enriched in surfaceantigens which are more likely to be relevant for immunotherapy and canbe repeatedly harvested from the same cells, thus reducing culturerequirements.

Finally, detergent treatment and ultracentrifugation were used toseparate tumor from Dr antigens. Much of the material shed by melanomacells is contained in fragments that are sedimented byultracentrifugation. Treatment with detergent breaks up these fragmentsso that most tumor antigens remain in suspension followingultracentrifuation, however, as found and confirmed by others, see thearticle by R. A. Reisfeld et al entitled "Approaches for the Isolationof Biologically Functional Tumor-Associated Antigens", Cancer Res1977;37:2860-2865, ultracentrifugation still causes sedimentation ofdetergent-treated transplantation antigens. This consequently provides asimple way of separating transplantation from tumor antigens.

The vaccine produced was found to be safe and immunogenic in 55 patientswith metastatic melanomas. There was no toxicity other than transienturticaria at the site of the injection. Immune responses to melanomawere induced or augmented in 64% of the patients. Cellular immunity wasinduced in 51% of the patients. The reaction seemed directed tomelanoma, since there was no response to concurrent skin tests withequal amounts of control vaccine prepared from pooled allogenicperipheral leukocytes or to human albumin. The cellular response wasinduced, rather than preexisting or due to an irritant in the vaccine,since there was no reaction to the initial injections of vaccine atdoses that subsequently led to strong skin reactions.

Humoral immunity to melanoma was increased in 24% of patients. Theresponses were generally weak; however, a major immunogenic contaminantin prior vaccine prepared from cultured cells, i.e. FCS proteins, seethe article by H. F. Seigler et al entitled "Specific ActiveImmunotherapy for Melanoma", Ann Surg; 1979;190:366-372, appears to havebeen successfully removed. No patient developed FCS antibodies, and byblocking studies with cold FCS, the melanoma antibodies induced in somepatients were not directed to FCS. The immunogenic antigens in thevaccine were surface proteins with MWs of about 75, 85, and 200+ Kd. Asdetermined by migration on SDS-PAGE, these were different from HLA or Drantigens, whose MW is <50 Kd.

Three of the 19 patients with Stage III disease did well followingimmunization. One had complete biopsy proven regression of metastaticcutaneous disease and remained healthy and free of melanoma 35 monthsafter starting immunization. Another remained disease-free followingresection of a solitary gall bladder metastases 13 months after startingvaccine therapy. The third patient showed a dramatic slowing ofpreviously rapidly progressing cutaneous disease following initiation ofvaccine immunotherapy of progression of cutaneous metastases resumedafter 6 months bvut the patient remained otherwise healthy and fullyactive 25 months after starting immunization.

These results are unexpected since immunotherapy is not thought to beeffective in patients with advanced dieases, see the article by R. K.Oldham et al entitled "Immunotherapy of the Old and the New", J BiolResponse Mod 1983;2:295-309. However, because the number of patients issmall and the course of melanoma is usually unpredictable, it isdifficult to judge the significance of these favorable responses inpatients with advanced melanoma.

In patients with less advanced Stage II melanoma, the vaccine appears tobe capable of slowing the progression of melanoma in some cases.Recurrences were lower and disease free interval was prolonged in 20 of45 patients with Stage II melanoma who developed a cellular immuneresponse to the vaccine. These differences were statisticallysignificant.

The availability of a characterized polyvalent melanoma antigen vaccinethat can be reproducibly made, safe to use, and immunogenic, shouldpermit a systematic study of the factors that influence theeffectiveness of active, specific immunotherapy of melanoma.

Other details of the practices of this invention described hereinaboveare to be found in the article by J. C. Bystryn et al entitled"Preparation and Characteristics of a Polyvalent Human Melanoma AntigenVaccine", Journal of Biological Response Modifiers 1986;5:211-224. Alsoof interest for more details of the practices of this invention is theto be published article by J-C Bystryn et al entitled "Immunogenecity ofa Polyvalent Melanoma Antigen Vaccine in Man", the article by M. Duganet al entitled "Relationship between Immune Responses to MelanomaVaccine Immunization and Tumor Regression in Man", Kaplan Cancer Center,NYU School of Medicine, New York, N.Y., published in American Federationfor Clinical Research, 1987 and the article by R. Oratz et al entitled"Induction of Lymphocytic Cell Infiltrate on Human Melanoma Nodules byActive Immunization to Melanoma Antigen Vaccine" to be published by AACR1987.

In the description of the invention set forth hereinabove, emphasis hasbeen placed upon the preparation of a vaccine useful for the preventionand/or treatment of melanoma. It is clearly indicated, however, that theconcept and practices of this invention are generally applicable to thepreparation of vaccines to treat or prevent human cancers and for thepreparation of vaccines to treat or prevent infectious diseases in manand animals.

The subject invention involves in a special embodiment, animmunotherapeutic approach for the prevention and/or treatment of cancerand infectious diseases. The administration to a patient of a vaccine inaccordance with this invention for the prevention and/or treatment ofcancer can take place before or after a surgical procedure to remove thecancer, before or after a chemotherapeutic procedure for the treatmentof cancer, and before or after radiation therapy for the treatment ofcancer and any combination thereof. It would appear, therefore, that thecancer immunotherapy in accordance with this invention would be apreferred treatment for the prevention and/or for the treatment ofcancer, particularly since the risk and side effects involved aresubstantially minimal compared with the other treatments, e.g. surgery,chemotherapy and radiation therapy. A unique aspect of this invention isthat the vaccines have the potential or capability to prevent cancer inindividuals without cancer but who are at risk of developing cancer.

The administration of a cancer vaccine prepared in accordance with thisinvention, is generally applicable to the prevention or treatment ofcancer. Cancers which could be suitably treated in accordance with thepractices of this invention include cancers of the lung, breast, ovary,cervix, colon, head and neck, pancreas, prostate, stomach, bladder,kidney, bone liver, esophagus, brain, testicle, uterus and the variousleukemias and lymphomas.

The vaccines in accordance with this invention, like the above-describedmelanoma vaccines, would be derived from the tumor or cancer cells to betreated. For example, in the treatment of lung cancer in accordance withthe practices of this invention, the lung cancer cells would be treatedas described hereinabove to produce a lung cancer vaccine. Similarly,breast tumor or cancer vaccine, colon cancer vaccine, pancreas cancervaccine, stomach cancer vaccine, bladder cancer vaccine, kidney cancervaccine and the like would be produced and employed as immunotherapeutcagents in accordance with the practices for the prevention and/ortreatment of the tumor or cancer cell from which the vaccine wasproduced.

Vaccines in accordance with this invention could, as stated, also beprepared to treat various infectious diseases which affect man andanimals by collecting the relevant antigens shed into the culture mediumby the pathogen. As there is heterogenecity in the type of immunogenicand protective antigens expressed by different varieties of organismscausing the same disease, polyvalent vaccines could be prepared bypreparing the vaccine from a pool of organisms expressing the differentantigens of importance.

In the practices of this invention set forth hereinabove for thetreatment of melanoma, the vaccine was intradermally or subcutaneouslyadministered to the extremities, arms and legs, of the patients beingtreated. Although this approach is generally satisfactory for melanomaand other cancers, including the prevention or treatment of infectiousdiseases, other routes of administration, such as intramuscularly orinto the blood stream may also be used. In addition, the vaccine can begiven togethe with adjuvants and/or immuno-modulators to boost theactivity of the vaccine and the patient's response.

The vaccines of this invention, as described hereinabove, have beenprepared from cells grown in flasks. The production of the vaccine can,if desired, be scaled up by culturing the cells in bioreactors orfermentors or other such vessels or devices suitable for the growing ofcells in bulk. In such apparatus the culture medium would be collectedregularly, frequently or continuously to recover therefrom the cell-shedor tumor-shed or materials or antigens before such materials or antigensare degraded in the culture medium.

If desired, devices or compositions containing the vaccine or antigensproduced and recoveed in accordance with this invention and suitable forsustained or intermittent release could be, in effect, implanted in thebody or topically applied thereto for the relatively slow release ofsuch materials into the body.

In summary, the essential features of vaccine preparation in accordancewith this invention are the following:

(a) The use of cultured cells to provide a continued and reproduciblesupply of material for vaccine production.

(b) The use of a pool of cell lines. Each line is selected on the basisof it expressing a different pattern of cell surface antigens. This isdone to ensure that the vaccine contains a broad representation ofdifferent antigens, hence, circumventing the problem antigenicheterogeneity of tumor cells.

(c) Adapting and maintaining the cells in serum-free medium. Thisexcludes from the vaccine undesirable and higly immunogeneic fetal calfserum proteins shown to be free of hepatitis, AIDS, and otherundesirable pathogens.

(d) Using shed material to obtain antigens for vaccine preparation. Thisprovides numerous advantages. It greatly simplifies the process ofcollecting bulk, on-line, production of antigens. The antigens which arecollected are solubilized. They are also partially purified as they areseparated from the bulk of cytoplasmic cellular components which areshed much more slowly. This greatly simplifies subsequent purificationor other biochemical procedures. Lastly, shed material permits theantigens to be harvested from the cells without cell destruction. Thisgreatly reduces the cost of cell culture requirements and hence the timeand cost of making the vaccine. The continued use of the same cells togenerate antigens also greatly reduces cell culture requirements.

The other steps in vaccine preparation can be individualized to satisfythe requirements of particular vaccines. In the case of melanomavaccines the shed material is concentrated and in some cases treatedwith detergent and ultracentrifuged to remove transplantationalloantigens.

The disclosures of all the above-identified reference publications areherein incorporated and made part of this disclosure.

As would be apparent to those skilled in the art in the light of theforegoing disclosure, many modifications, alterations and substitutionsare possible in the practices of this invention without departing fromthe spirit or scope thereof.

                  TABLE 1                                                         ______________________________________                                        MAA immunophenotyping of melanoma vaccine                                                MAA       Presence of MAA                                                     defined   (vaccine batch)                                          Antisera     (kilodaltons)                                                                             1      2    3    Ref.                                ______________________________________                                        Mouse monoclonal                                                              225.28S      240+        +      +    +    23                                  9.2.27       240+        +      +    +    24                                  436.G10      122-130     0      .sup. NT.sup.b                                                                     NT                                       Nu4B          26, 29, 95, 116                                                                          +      NT   NT   25                                  376.96        94         0      NT   NT   17                                  118.1         94-97      +      +    +    15                                  465.12S       94         0      NT   NT   27                                  MeTBT         69-70      0      NT   NT   26                                  Rabbit polyclonal                                                             SB29, SB54   240         +      +    +    11                                  SB29, SB54   150.sup.a   +      +    +    11                                  SB29, SB54   140.sup.a   +      +    +    11                                  SB29, SB54   120         +      +    +    11                                  SB29, SB54    95         +      +    +    11                                  SB29, SB54    75         +      +    +    11                                  ______________________________________                                         .sup.a Not reactive with SB54.                                                .sup. b (NT) not tested.                                                 

                  TABLE 2                                                         ______________________________________                                        Effect of detergent and ultracentrifugation on macromolecules,                MAAs, and Dr antigens in material shed by melanoma cells                      Presence in shed material after ultracentrifugation                           .sup.125 I-macro-                                                             molecules.sup.b  .sup.125 I-MAAs.sup.c                                                                     .sup.125 I-Dr.sup.c                                              Change.sup.d  Change      Change                              Treatment.sup.a                                                                       cpm     (%)      cpm  (%)    cpm  (%)                                 ______________________________________                                        None    10,362           817         428                                      Ultracentri-                                                                          6,325   -40      258  -70     0   -100                                fugation                                                                      NP-40 + 8,612   -17      574  -30     0   -100                                ultracentri-                                                                  fugation                                                                      ______________________________________                                         .sup.a Material shed by radioiodinated melanoma cells was ultracentrifuge     in 0.5 ml aliquots at 100,000 g for 90 min, incubated in a final              concentration of 0.5% NP40 for 2 h prior to ultracentrifugation, or not       treated. All were subsequently assayed for radioactivity associated with      macromolecules, MAAs defined by antiserum SB29, or Dr antigens. All assay     were performed on 0.025 ml aliquots of material in the presence of a fina     concentration of 0.5% NP40.                                                   .sup.b Assayed by precipitation with 10% trichloroacetic acid.                .sup.c Assayed by protein Aimmune precipitation with specific antisera.       .sup.d From untreated control.                                           

                                      TABLE 3                                     __________________________________________________________________________    Surface MAAs expressed by melanomas in various individuals                    __________________________________________________________________________              Expression of MAA in melanoma.sup.a                                 MAA Antiserum                                                                           HM31  HM34 HM49 HM54 HM60  HM80 G361 SK23 SK27  SK28                __________________________________________________________________________    240+                                                                              SB29  +     +    -    ++   +++   -    +++  ++   ++    ++                      SB54  +     +    -    +    -     -    ++   -    -     -                       225.28S                                                                             -     -    -    -    +++   +++  -    +    -     +++                     9.2.27                                                                              -     -    -    -    +++   +++  -    +    ±  +++                 150 SB29  +     +    +    +    -     -    +    -    -     -                   140 SB29  ++    +    +    +++  -     -    +++  -    -     -                   120 SB29  +++   ++   +    +++  -     -    +++  -    -     -                       SB54  ++    +    +    ++   -     -    ++   -    -     -                   116 Nu4B  -     -         -    -     -    -    -    -     -                    95-97                                                                            SB29  ++    +    -    +++  +     -    +++  +    +     -                       SB54  ++    +    -    +++  -     -    +++  -    -     -                       118.1 -     -    -    -    ++    +++  -    +++  +++   +++                  75 SB29  ++    -    -    +++  +++   +++  +++  +++  +++   +++                     SB54  +     -    -    +    -     -    +    +    -     -                    70 Me3 TBT                                                                             -     -         -    -     -    -    -    -                         __________________________________________________________________________                                         Expression of MAA in melanoma.sup.a                                 MAA Antiserum                                                                           SK29 SK37 M14  M20   VAL                 __________________________________________________________________________                               240+                                                                              SB29  +    +    ++   ++    ++                                                 SB54  -    -    -    -     -                                                  225.28S                                                                             +++  +++  +++  +++   -                                                  9.2.27                                                                              +++  +++  +++  +++   -                                              150 SB29  -    -    ++   -     -                                              140 SB29  -    -    -    -     -                                              120 SB29  -    -    -    -     -                                                  SB54  -    -    -    -     -                                              116 Nu4B  +    -    -    +     -                                               95-97                                                                            SB29  -    -    -    -     +                                                  SB54  -    -    -    -     -                                                  118.1 -    +++  ++   +++   +++                                             75 SB29  ++   +    ++   ++    +++                                                SB54  -    -    -    -     -                                               70 Me3 TBT                                                                             -    -    -    -     -                   __________________________________________________________________________     .sup.a Assayed by indirect immunoprecipitation with protein Asepharose.  

                                      TABLE 4                                     __________________________________________________________________________    Characteristics of immunized patients                                                   Previous      Duration of metastatic Length of                      Patient   treatment other                                                                       Site of                                                                             disease prior to                                                                         No. of Current                                                                            follow-up.sup.b                no. Age                                                                              Sex                                                                              than surgery                                                                          metastasis                                                                          immunization (months)                                                                    immunization                                                                         status.sup.a                                                                       (months)                       __________________________________________________________________________    1   31 F  BCG,.sup.c DTIC                                                                       Skin, lung                                                                          12         10     P    1/4                            2   24 F  None    Lung  2          10     P    3                              3   53 M  None    Skin  2          13          8                              4   58 F  None    Skin  2           8     P    1                              5   48 M  None    Skin  1          12     P    3                              6   54 M  None    Skin  2          11     P    3                              7   58 M  None    Skin  2          14     P    4                              8   68 M  None    Skin, lung                                                                          2          10     P    6                              9   75 F  DTIC,   Skin  36         17     S    14                                       Actinomycin D                                                       10  46 M  None    Skin  1          18     R    24                             15  29 M  None    Skin  4           8     P    2                              17  68 M  None    Skin  4           8     P    2                              20  38 M  None    Skin, lung                                                                          3          13     P    4                              __________________________________________________________________________     .sup.a (P) progression; (S) stable; (R) regression.                           .sup.b From onset of immunotherapy.                                           .sup.c (BCG) bacillus CalmetteGuerin.                                    

                  TABLE 5                                                         ______________________________________                                        Immunogenicity of melanoma vaccine                                            Patient      Immune response to melanoma.sup.a                                no.          Humoral.sup.b                                                                             Cellular.sup.c                                                                         Either                                      ______________________________________                                        1            ++          0        5                                           2            +           10       +                                           3            +           0        +                                           4            0           0        0                                           5            ±        5        0                                           6            0           0        0                                           7            0           0        +                                           8            0           0        0                                           9            0           20       +                                           10           +           10       +                                           15           ++          0        +                                           17           0           NT       0                                           20           NT          25       +                                           No. (%) positive:                                                                          5 (38%)     4 (31%)  8 (62%)                                     ______________________________________                                         .sup.a (±) 25-49%, (+) 50-100%, and (++) 100%, increase over               preimmunization level of melanoma antibodies; (NT) not tested.                .sup.b By indirect immunoprecipitation of .sup.125 Imelanoma                  macromolecules.                                                               .sup.c By skin test to 10 μg vaccine; results represent millimeters of     average induration at 24-48 h.                                           

                  TABLE 6                                                         ______________________________________                                        Antibodies to fetal calf serum proteins                                       in patients immunized to melanoma vaccine                                     Patient    Antibodies to                                                                              .sup.125 I-FCS.sup.a (2 months                        no.        preimmunization                                                                            postimmunization)                                     ______________________________________                                        Melanoma                                                                        1        18.3         0.7                                                     2        0.0          0.1                                                     3        0.0          0.0                                                     4        0.1          0.1                                                     5        0.1          0.2                                                     6        0.1          <0.1                                                    7        <0.1         0.1                                                     8        <0.1         <0.1                                                    9        0.6          0.8                                                    10        0.0          0.0                                                    15        0.1          <0.1                                                   17        0.3          0.4                                                    20        0.0          0.0                                                   Normal                                                                        2003       0.0                                                                2004       0.1                                                                2005       <0.1                                                               2006       0.0                                                                2007       0.0                                                                2008       0.0                                                                2009       0.0                                                                2010       0.1                                                                2011       0.0                                                                2012       0.0                                                                2013       0.0                                                                ANTI-FCS   68.0                                                               ______________________________________                                         .sup.a Percent of radioactivity associated with .sup.125 IFCS specificall     immunoprecipitated by serum and protein Asepharose.                      

What is claimed is:
 1. A method for preparing a polyvalent humanmelanoma vaccine comprising:(a) culturing over a period of about 3 hoursin serum free medium a pool of human melanoma cell lines, wherein saidcell lines are selected based on the shedding of different molecularweight cell surface melanoma associated antigens, said melanoma cellsprior to culturing having been adapted to and maintained in serum freeculture medium, (b) subjecting the culture medium, after culturing themelanoma cells therein, to a particle separation operation for theremoval of melanoma cells from said culture medium, (c) concentratingthe resulting melanoma cell free medium which contains shed melanomaassociated cell surface material therein shed from said melanoma celllines during culturing; and (d) utilizing said recovered shed antigenmaterial in the preparation of said vaccine comprising said shedmelanoma associated cell surface antigens.
 2. A method in accordancewith claim 1 wherein said melanoma cell lines are selected on the basisof the shedding of different patterns of shed melanoma associated cellsurface antigens in the molecular weight range from 30 kD to 240 kD. 3.A method in accordance with claim 1 wherein said vaccine is stored at alow temperature prior to use.
 4. A method in accordance with claim 1wherein said particle separation operation comprises centrifugation. 5.A method in accordance with claim 1 wherein an adjuvant or nonionicmodulator is added to said vaccine.